The present invention relates to a method for the determination of non-, anti-, or pro-apoptotic and necrotic conditions of cells, using newly designed vectors coding for marker proteins, cell lines transfected with such vector, and a method to assay the non-, pro-, or anti-apoptotic or necrotic activity of test compounds or physical stimuli.
Apoptosis plays an essential role in development, i.e. embryogenesis and normal cell turnover, but also in diseases such as cancer, AIDS, neurodegeneration and viral infections. Unlike necrosis, apoptosis is an active, gene-directed self-destruction process of the cell and is associated with characteristic morphological and biochemical changes1,2. Nuclear and cytoplasmic condensation and fragmentation of the dying cell into membrane-bound apoptotic bodies are typical characteristics of apoptosis. Another feature of apoptotic cell death is the chromosomal DNA degradation into oligonucleosomal fragments after the activation of specific nucleases3,4.
Apoptosis can be induced by the interaction of the cell surface molecule Fas (CD95) with Fas-Ligand (FasL), where the Fas expressing and sensitive cells undergo apoptosis. Fas is a type I membrane protein, which belongs to the tumor necrosis factor (TNF) and nerve growth factor (NGF) receptor family5-7. Fas expression is found on a wide variety of tissues and cells such as thymus, liver, lung, ovary, heart and myeloid cells9-12. FasL expression is not only found on lymphocytes but also on a wide variety of tissues and some tumors13-19. Both membrane bound FasL and the soluble form (sFasL) can induce apoptosis in Fas positive and sensitive cells. Other forms among a variety of apoptosis mediators include the Perforin/Granzyme system, the TRAIL/TRAIL-R system36, cytokine deprivation (e.g. IL-3 deprivation) and irradiation.
In contrast to apoptosis, necrosis is a non-physiological death of cells due to chemical or physical injury of the cell membrane. Morphological criteria include cell swelling and cell lysis, lysosomal leakage and loss of the cell membrane integrity.
During the last decade, it has become clear that apoptosis plays a keyrole in several diseases. Apoptosis is increased in AIDS, but decreased in cancer and certain autoimmune proliferative diseases.
Flow cytometry offers a wide variety of possibilities to measure apoptosis. Different methods have been established and implemented, some which stain on the cell surface and some which stain intracellularily.
One of the first approaches was, beside the observation that apoptotic cells shrink and have higher intracellular granularity, to stain with DNA specific fluorochromes (e.g. propidium iodide [PI], ethidium bromide [EtBr]). As soon as a lethal hit is being induced, the DNA starts. to change its profile. Apoptotic DNA not only consists of fragmented DNA (visualised as shorter bands, so called DNA ladder, in an agarose gel) but is also partially digested into single nucleotides, so that fluorochromes, like PI or EtBr, have less DNA to stain. This is typically observed by a shift to the left, called sub-G1 peak, on the specific fluorochrome detection channel in the FACScan(trademark) (from Becton Dickinson, USA). The big disadvantage of this method is that the cell membranes have first to be permeabilized with reagents, like ethanol, in order to stain them with DNA specific dyes, like PI20. The treatment is time and labour consuming, and the risks of loosing cells and of handling errors are high. Furthermore, the discrimination between live and apoptotic cells cannot be standardised and requires large experience.
Another method is the terminal deoxynucleotidyl transferase (TdT)-mediated endlabeling of the DNA strand breaks (TUNEL). The TUNEL method detects DNA strand breaks in cells undergoing apoptosis. TdT is an enzyme which catalyzes the addition of deoxyribonucleotide triphosphate to the 3xe2x80x2-OH ends of double or single-stranded DNA. Unlike normal cells, apoptotic cell nuclei incorporate exogenous nucleotides (dUTP)-DIG in the presence of TdT. An anti-DIG antibody fragment with a conjugated fluorochrome enables the visualisation of apoptotic cells. An increase of apoptotic cells causes a higher number of DNA fragments and consequently a brighter fluorescence. An advantage of this method is the very high specificity21. A disadvantage of this method is that it is expensive and can only be used for a small set of samples, because it is time intensive. Therefore, it is not applicable for large screening programmes.
The loss of cell membrane polarity and the presentation of increased amounts of phosphatidyl serine (PS) on the outside of the cell membrane during the early phase of apoptosis has led to yet a new approach. Annexin V is a calcium-dependent phospholipid binding protein with high affinity for PS. Due to the fact, that the cell membrane integrity is maintained in the early and intermediate phases of apoptosis but not in necrosis, it is possible to distinguish between apoptotic and necrotic cells, when Annexin V is used concomitantly with the DNA dye PI. Early and intermediate apoptotic cells show increased binding of Annexin-FITC and are mainly negative for PI-staining. Late apoptotic stages and necrotic cells become double positive, because of PS presentation on the surface and the PI staining of intracellular nucleic acids due to disintegration of the membrane22. This method is also costly and labour intensive.
Green fluorescence protein (GFP) from the jellyfish Aequorea victoria can be used to monitor gene expression and protein localization in living organisms (in vivo) and in vitro23-26. GFP-fluorescence is stable, can be monitored noninvasively in living cells and persists in paraformaldehyde-fixed cells. FACS-optimized mutants of green fluorescence protein have been developed8. One of these mutants (GFPmut1) has been integrated into the PEGFP vectors and is commercially available (from Clontech). The big advantage of this mutant is that the maximal excitation peak of GFPmut1is 488 nm and the emission maxima is 507 nm. Conventional flow cytometers are equipped with an argon laser emitting light at 488 nm and have the suitable detection filters already installed, making the GFPmut1-protein an ideal candidate for flow cytometry studies and fluorescence microscopy.
GFP has already been used as marker for visualizing changes in cell morphology such as blebbing caused by cytotoxic agents or apoptosis, or as transfection marker (WO97/11094), or as marker for screening factors modulating gene expression (WO97/14812). GFP has also been used as a marker protein to detect the progression of the morphological changes of apoptotic cells37.
GFP has been used as a marker protein to detect cells transiently transfected with the commercially available plasmid pEGFP-C1 (Clontech)34. According to Lamm et al.34 apoptosis was detected by reduced fluorescence of the DNA-binding dye PI in the apoptotic subpopulation. It was not recognized that GFP itself could be used as a marker for apoptosis. The great disadvantage of PI-staining is that no changes in the state of one and the same cells can be monitored but only one specific state since for PI-staining the cells have to be permeabilized and fixed.
Experiments by the present inventors have shown, that stable transfection of eukaryotic cells (e.g. A20.2J) with the pEGFP-C1 (as received from the manufacturer Clontech with the cytomegalovirus CMV promoter) used in the above cited state of the art for tansient transfection results in little or no expression of the GFPmut1gene.
For the foregoing reason there is a need for a new and improved method and tools for determining apoptosis and necrosis of cells which in particular can be used to efficiently and cheaply assay compounds on their pro-apoptotic or anti-apoptotic or necrotic activity.
Accordingly, objects of the invention are to provide an improved method for the determination of apoptosis and/or necrotic conditions of cells and to provide improved tools useful for this method.
The present invention is directed to a method for the determination of apoptotic and/or necrotic conditions of living test cells that satisfies the hereinbefore discussed needs. This method comprises monitoring changes in the signal or the intensity, respectively, of a marker protein in said test cells, in particular a method wherein the apoptotic and/or necrotic condition is monitored in the presence of a non-, pro- or anti-apoptotically or necrotically active compound, and/or a physical stimulus, and wherein the marker protein is preferrentially produced in the test cells after stable transfection of said cells with a DNA coding for and expressing the marker protein.
Preferred marker proteins are fluorescent marker proteins with the Green Fluorescent Protein (GFP) or a fluorescent mutant thereof, e.g. GFPmut1, being particularly preferred.
Another object of the present invention is a new vector for the transfection, preferably the stable transfection of test cells which are able to highly express said marker protein, preferably a fluorescent protein, in particular GFP or a mutant thereof. It was found that the known hEF-1xcex1 promoter, and the new combination of the CMV and the MoLV-LTR promoter are particularly well suited for enhancing transcription of the GFPmut1 gene.
Another object of the present invention are live cells or live cell lines, respectively stably transfected with such a vector. Such a cell line can be used in an assay to determine non-, pro- or anti apoptotic or necrotic activity of test compounds. Although it is possible to transfect live cells with more than one vector with different marker molecules, for e.g. the simultaneous detection of different signals, usually one marker is sufficient.
Another object of the present invention is a method to assay the non-, pro or anti-apoptotic or necrotic activity of a test compound on live test cells. This method comprises stably transfecting a group of said cells with at least one vector coding for and expressing a marker protein. The transfected cells are treated in a suitable culture medium with a test compound. The change in the signal or intensity of a signal, respectively, i.e. the decrease or increase, of the expressed marker protein in said group of cells is monitored by conventional methods, and compared with the results observed with a parallel group of the same test cells which was not exposed to the test compound but otherwise identically treated. The test compound may consist of a multiplicity of compounds, e.g. as obtained from combinatorical chemistry methods. Of particular interest are the apoptotic or necrotic conditions of normal and cancer cells under the influence of test compounds and/or physical stimuli.
In a specific embodiment of the methods of the present invention the test cells or cell line are transfected with a test gene expressing a protein of interest such as an apoptosis inhibitor or an apoptosis stimulator.
These and other features, aspects, and advantages of our invention will become better understood with reference to the following description of modes for carrying out the invention, the appended claims and accompanying drawings.